Tissue-Specific Promoters for the Generation of Transgenic Plants
The recent advancements in plant transformation techniques offer new opportunities to the improvement of crops. Following the transgenic approach, new characters can be introduced in the plants, which contribute to the increase of plant productivity, product quality and to improve the resistance of plants to adverse climatic conditions as well as to pathogens. In addition, transgenic plants can be used to produce recombinant proteins, biopolymers, medicaments, vaccines or antibodies (L. Lanfranco, Riv Biol. 2003, 96:31-54; Dunwell J M, J. Exp. Bot., 2000, 51:487-496).
The production of recombinant proteins in plants requires the use of promoters able to direct the correct expression of transgenes in vegetal tissues. To date, a limited number of promoters have been proposed for use in the generation of transgenic plants. Most of them are constitutive promoters, such as the 35S promoter from the cauliflower mosaic virus (CaMV35S) (Odell et al., Nature, 1985, 313:810-812) or the ubiquitin promoter (Holtorf et al., Plant Mol. Biol., 1995, 29:637-646).
A drawback of such promoters is that they are active in nearly all the plant tissues, thus preventing selective transgene expression in specific organs or during particular growth stages of the transgenic lineage, unlike tissue-specific promoters, which direct the production of recombinant proteins in selected tissues or organs. For example, the promoters involved in the accumulation of spare substances in seeds, such as phaseolina (Bustos et al., Plant Cell, 1989, 1:839-853) or 2S albumin (Joseffson et al., J. Biol. Chem., 1987, 262:12196-12201), direct the seed-specific expression of transgenes. The Rubisco small subunit promoter or the potato ST-LSI promoter direct leaf-specific transgene expression (Stockhouse et al., EMBO J., 1989, 8:2445). Although many other tissue- or organ-specific promoters have been described in the literature, only few of them show selectivity for a determined plant cell-type. These promoters should direct transgene expression limited to particular cells within the plant organ.
Stomata: Anatomy and Function
Stomata are small apertures present on the surface of aerial organs of land plants. These structures play an important role in the regulation of gas fluxes between the plant tissues and the atmosphere, allowing either CO2 influx, which is necessary for the photosynthesis, or water loss by transpiration. The stoma consists of two highly specialized epidermal cells, called guard cells, the movement of which determines the opening/closure of the stomatal rima (FIG. 1).
The level of stomatal opening reflects the balance between the need of CO2 for the photosynthesis and water availability. Thus, it is not surprising that land plants have developed complex regulation mechanisms modulating the stomatal opening/closing process in response to environmental stimuli or to endogenous signals (Wilmer and Fricker, 1996, Stomata, Ed Chapman and Hall, London, 1-375).
The guard cell shape is determined by volume changes induced by turgor modifications. The latter are in turn induced by the exchange of solutes, either inorganic, such as K+ and Cl−, or organic, such as saccharose or malate, in the cell lumen (Schroeder et al., Ann. Rev. Plant Physiol. Plant Mol. Biol., 2001, 52:627-6658). Conditions favouring the photosynthetic activity, such as the presence of light and of elevated CO2 concentrations, promote the accumulation of solutes in the guard cells, whereby an increased turgor induces stomatal opening (FIG. 1A).
On the contrary, in the absence of water, the phytohormon abscisic acid (ABA), induces a rapid diminution of guard cell turgor, resulting from the efflux of K+, Cl− and saccharose and from the conversion of malate into osmotic-inactive starch, thereby causing stomatal closure (FIG. 1B).
The reduction of stomatal aperture, mediated by ABA accumulation, represents the main adaptive response of plants to drought, allowing to minimize the loss of water by transpiration (Wilkinson and Davies, Plant Cell Env., 2002, 25:195-210). Recently, many components of the ABA signal transduction cascade have been identified in guard cells following a pharmacological or genetic approach.
The ABA-induced stomatal closure involves the increase of Ca++ cytosolic concentration, the activation of anion channels, the modification of cytoplasmic pH and of potassium channel activity, the production of oxygen reactive molecules, the regulation of phosphatases and kinases and of other proteins such as heterotrimeric G-proteins, farnesyltransferase and mRNA cap-binding protein (Schroeder et al, Ann. Rev. Plant Physiol. Plant Mol. Biol., 2001, 52:627-6658).
The modulation of hormon signal transduction mechanisms, having a direct influence on stomatal opening/closure, provides a valuable tool for the generation of crop plants resistant to adverse environmental or climatic conditions, expecially to drought, and in which the exchange of CO2, and therefore the photosynthetic process, is optimized.